Mouse monoclonal antibodies to Hepatitis E virus and methods for using same

ABSTRACT

Monoclonal antibodies are described which specifically bind to Hepatitis E Virus (HEV), and more particularly to HEV orf-3 antigen. Also provided are hybridoma cell lines which secrete these monoclonal antibodies, methods for using these monoclonal antibodies, and assay kits which contain these monoclonal antibodies.

This application is a Continuation of application Ser. No. 08/492,606,filed Jun. 20, 1995 abandoned, which is a Continuation of applicationSer. No. 08/172,700, filed Dec. 22, 1993 abandoned.

BACKGROUND OF THE INVENTION

This invention relates generally to antibodies which specifically bindto Hepatitis E Virus (HEV), and more particularly, relates to a novelhybridoma cell line which secretes monoclonal antibodies to HEV off-3antigen, and methods for using these monoclonal antibodies.

HEV, variously referred to as waterborne, epidemic or entericallytransmitted non-A, non-B hepatitis (ET-NANBH), has a global distributionand has been noted as the cause of major endemic outbreaks of viralhepatitis in developing countries. D. W. Bradley et al., Br. Med. Bull.46:442-461 (1990). Sporadic cases of ET-NANBH, as well as importedtravel exposure, have been reported in developed countries. S. J.Skidmore et al., Lancet 337:1541 (1991). Although the fecal-oral routeof transmission predominates, limited person-to-person routes ofexposure have been suggested in some epidemiological studies. O.Velasquez et al., J. Amer. Med. Assoc. 363:3281-3285 (1990). Thisdisease has been documented as having a high mortality rate ofapproximately 20% in pregnant women infected during their thirdtrimester of pregnancy. See D. W. Bradley et al., supra.

Molecular cloning of the putative agent of HEV has been hampered by thelack of a tissue culture system for virus propagation. However, the useof available animal models and a newly developed non-specificamplification procedure have allowed the identification of a unique cDNAclone (identified as "ET 1.1") obtained from bile of cynomolgus macaquesinfected with a Burmese strain of HEV. A. G. Andjaparidze et al., Vopr.Virusol. 1:73-80 (1986), D. W. Bradley et al., Proc. Natl. Acad. Sci.USA 84:6277-6281 (1987) and G. W. Reyes et al., Science 247:1335-1339(1990). Successful confirmation of the viral origin of this clone led tothe identification of similar sequences in human fecal specimenscollected from ET-NANBH epidemics in Somalia, Tashkent, Borneo, Pakistanand Mexico. G. R. Reyes et al., supra. cDNA libraries also have beenprepared from human stool samples obtained during an ET-NANBH outbreakin Mexico. G. R. Reyes et al., Gastroenterol. Japon. 26:142-147 (1991).Immunoscreening of these cDNA libraries led to the identification of twocDNA clones which encode epitopes specific for HEV. P. O. Yarbough etal., J. Virol. 65:5790-5797 (1991). The isolation and sequencing of aset of overlapping cDNA clones led to the recognition that this form ofhepatitis is caused by a novel virus unlike any of the other molecularlycharacterized agents of viral hepatitis. A. W. Tam et al., Virology185:12-131 (1991).

Various regions of the HEV genome have been cloned and expressed in E.coli as fusion proteins with glutathione-S-transferase (GST). See, forexample, S. J. Skidmore et al., supra. Four of these recombinantantigens, two derived from a Burmese (B) strain of HEV and two derivedfrom a Mexican (M) strain of HEV, have been shown to contain antigenicsites recognized by antibodies from individuals previously exposed toHEV. See, P. O. Yarbough et al., supra. The two antigens from theMexican strain, named M3-2 and M4-2, correspond to amino acid sequencesat the carboxy-terminus of the second open reading frame (ORF-2) and thethird open reading frame (orf-3), respectively. The two antigens fromthe Burmese strain, B 3-2 and B 4-2, correspond to amino acid sequencesat the carboxy-terminus of orf-2 and orf-3, respectively. The M 3-2 andB 3-2 recombinant antigens both comprise 42 amino acids from the carboxyterminus of ORF-2. The degree of amino acid homology between thesesequences of 42 amino acids is 90.5%. Id. The M 4-2 and B 4-2recombinant antigens each comprise 33 amino acids from the carboxyterminus of orf-3; the degree of homology between these two sequences of33 amino acids is 73.5%. Id.

Tests developed for detection of HEV must contain reagents which areuseful for determining the specific presence of the virus in a testsample. The need therefore exists for reagents, such as monoclonalantibodies, capable of reacting only with HEV. Additionally, the abilityto produce pure, specific monospecific antibodies is clearly of greatimportance for accurate identification, characterization, andpurification of the HEV antigen.

While methods are available to confirm screening assay results foragents such as HIV, these techniques are not yet available forconfirming the presence of HEV. Methods such as culturing HEV in vitroand a viral-based Western blot test are not available. While thedetection of HEV nucleic acid may be done by performing PCR, thistechnique is tedious and expensive, requires special equipment such as athermocycler, and turn-around time is up to 24 hours. Immunoelectronmicroscopy (IEM) has been used to confirm the presence of anti-HEVantibody, but the use of IEM is cost prohibitive as a routineconfirmatory tool.

It therefore would be advantageous to provide a monoclonal antibodywhich can be used in accurate, rapid and cost effective methods forscreening for HEV antigens or HEV antibodies in a test sample.

SUMMARY OF THE INVENTION

The present invention provides a highly specific and novel monoclonalantibody or fragment thereof that can be employed for the detection ofHEV orf-3 antigen. The monoclonal antibody specifically binds to the HEVorf-3 protein at the carboxy terminal. The hybridoma which secretes themonoclonal antibody is identified as: Hybridoma cell line H67C46(A.T.C.C. deposit No. HB 11522, secreting monoclonal antibody (H67C46).The specificity of this monoclonal antibody enables the advantageousidentification of HEV-orf-3 antigen, which identification can be usefulin viral differentiation studies as well as in the diagnosis andevaluation of HEV infections.

The monoclonal antibody of the present invention can be used in animmunoassay for the detection of HEV antigen or antibody. Many suchimmunoassay formats are known in the an and can be modified by adding aknown amount of a monoclonal antibody which specifically binds to HEVorf-3 antigen.

A particularly preferred assay format is a competitive assay fordetermining the presence and amount of HEV antibody which may be presentin a test sample. The assay involves contacting a test sample suspectedof containing HEV antibodies with a solid phase coated with HEV antigensand an indicator reagent comprising a signal generating compound and amonoclonal antibody which specifically binds to HEV orf-3 antigen, for atime and under conditions sufficient to form antigen/antibody complexesof the test sample and solid phase and/or indicator reagent and solidphase; and determining the presence of HEV antibody present in the testsample by detecting the reduction in binding of the indicator reagent tothe solid phase as compared to the signal generated from a negative testsample to indicate the presence of HEV antibody in the test sample

Assay kits which contain the monoclonal antibody of the presentinvention are also described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the plasmid used for productionof orf-3 CKS/HEV 8-5 antigen from plasmids pJO201 and pGEX-HEV-ORF3-8-5.

DETAILED DESCRIPTION OF THE INVENTION

The monoclonal antibody of the present invention can be employed invarious assay systems to determine the presence, if any, of HEV antigensor HEV antibodies in a test sample. Fragments of the monoclonal antibodyprovided also may be used.

The monoclonal antibody of the present invention may be screened for asfollows. Recombinant or synthetic HEV off-3 antigen is used on a solidphase (preferably, polystyrene beads). For example, the CMP-KDOsynthetase (CKS) HEV recombinant off-3 (8-5), obtained as described inExample 1, below is coated on the solid phase in the unpurified(extracted and solubilized) form. Alternately, an immunoassay (EIA)which uses the synthetic peptide spB4-2 (obtained as described incopending application U.S. Ser. No. 08/089,877 filed Jul. 9, 1993) onthe solid phase is utilized. Detection of non-specific binding isaccomplished by coating the solid phase with CKS alone or with a non-HEVCKS recombinant protein. Test samples (mouse serum, tissue culturesupernatant, or mouse ascites fluid) are serially diluted in a specimendiluent and a portion is incubated with each solid phase for 1-2 hoursat 40° C. Beads are then washed with buffer and bound antibody isdetected using horseradish peroxidase (HRPO)-labeled second antibody.Beads are incubated with conjugate for a sufficient time at 40° C., andwashed as before. An appropriate substrate solution is added in a darkat room temperature for 30 minutes. Sulfuric acid is added to stop thereaction, and the amount of color generated is determined by measuringthe absorbance of the substrate at 492 nm within 2 hours of sulfuricacid addition. Details of this method are described in the examplesection, below.

For example, in a preferred assay format, the monoclonal antibody of theinvention can be employed as a competitive probe for the detection ofantibodies to HEV antigen. For example, HEV antigens coated on a solidphase, are contacted with a test sample suspected of containing antibodyto HEV, and incubated with an indicator reagent comprising a signalgenerating compound which generates a measurable signal attached to themonoclonal antibody of the invention for a time and under conditionssufficient to form antigen/antibody complexes of the test sample to thesolid phase or the indicator reagent to the solid phase. The reductionin binding of the monoclonal antibody of the invention to the solidphase, as evidenced by a reduction in the generated signal, can bequantitatively measured. A measurable reduction in the signal comparedto the signal generated from a confirmed negative HEV test sample wouldindicate the presence of anti-HEV antibody in the test sample.

In an alterative assay method for detection of HEV antigens, apolyclonal or monoclonal anti-HEV antibody or a fragment thereof, whichhas been coated on a solid phase, is contacted with a test sample whichmay contain HEV antigens, to form a mixture. This mixture is incubatedfor a time and under conditions sufficient to form antigen/antibodycomplexes. Then, an indicator reagent comprising a monoclonal or apolyclonal antibody or a fragment thereof, which specifically binds toHEV antigen, to which a signal generating compound which generates ameasurable signal has been attached, is contacted with theantigen/antibody complexes to form a second mixture. This second mixturethen is incubated for a time and under conditions sufficient to formantibody/antigen/indicator reagent complexes. The presence of HEVantigen present in the test sample and captured on the solid phase, ifany, is determined by detecting the measurable signal generated by thesignal generating compound. The amount of HEV antigen present in thetest sample is proportional to the signal generated.

Attentively, a polyclonal or monoclonal anti-HEV antibody or fragmentthereof which is bound to a solid support, the test sample and anindicator reagent comprising a monoclonal or polyclonal antibody orfragments thereof, which specifically binds to HEV antigen to which asignal generating compound which generates a measurable signal isattached, are contacted simultaneously to form a mixture. This mixtureis incubated for a time and under conditions sufficient to formantibody/antigen/indicator reagent complexes. The presence, if any, ofHEV antigen present in the test sample and captured on the solid phaseis determined by detecting the measurable signal generated by the signalgenerating compound. The amount of HEV antigen present in the testsample is proportional to the signal generated. In this or the assayformat described above, the monoclonal antibody of the invention can beemployed either as the capture phase or as pan of the indicator reagent.

In yet another detection method, the monoclonal antibody of the presentinvention can be employed in the detection of HEV antigen in fixedtissue sections, as well as fixed cells by immunohistochemical analysis,by standard methods well-known to those skilled in the art.

In addition, the monoclonal antibody can be bound to mantrices similarto CNBr-activated sepharose and used for the affinity purification ofspecific HEV antigens from cell cultures, or biological tissues such asblood and liver.

The monoclonal antibody of the invention also can be used for thegeneration of chimeric antibodies for therapeutic use, or other similarapplications.

The monoclonal antibody or fragment thereof can be provided individuallyto detect HEV antigen. Combinations of the monoclonal antibody (andfragments thereof) of the present invention provided herein also may beused in combination with other monoclonal antibodies that have differingspecificities for HEV as components in a mixture or "cocktail" of HEVantibodies, each having different binding specificities. Thus, thiscocktail can include the monoclonal antibody of the invention directedto off-3 protein from the HEV genome, along with different monoclonalantibodies directed to other HEV regions, such as the off-2 protein orother binding sites on orf-3 HEV proteins. This cocktail of monoclonalantibodies would then be used in place of the single monoclonal antibodyas described in the assay formats herein.

The polyclonal antibody or fragment thereof which can be used in theassay formats should specifically bind to HEV antigen. The polyclonalantibody used preferably is of mammalian origin and includes but is notlimited to human, goat, rabbit or sheep anti-HEV polyclonal antibody.The polyclonal antibodies used in the assays can be used either alone oras a cocktail of polyclonal antibodies. Since the cocktails used in theassay formats are comprised of either monoclonal antibodies orpolyclonal antibodies having different HEV specificity, they would beuseful for diagnosis, evaluation and prognosis of HEV infection, as wellas for studying HEV protein differentiation and specificity.

Test samples which can be tested by the methods of the present inventiondescribed herein include human and animal body fluids such as wholeblood, serum, plasma, cerebrospinal fluid, urine, biological fluids suchas cell culture supernatants, fixed tissue specimens and fixed cellspecimens.

The "solid phase" is not critical and can be selected by one skilled inthe art. Thus, latex particles, microparticles, magnetic or non-magneticbeads, membranes, plastic tubes, walls of microliter wells, glass orsilicon chips and tanned sheep red blood cells are all suitableexamples. Suitable methods for immobilizing peptides on solid phasesinclude ionic, hydrophobic, covalent interactions and the like. A "solidphase", as used herein, refers to any material which is insoluble, orcan be made insoluble by a subsequent reaction. The solid phase can bechosen for its intrinsic ability to attract and immobilize the capturereagent. Alternatively, the solid phase can retain an additionalreceptor which has the ability to attract and immobilize the capturereagent. The additional receptor can include a charged substance that isoppositely charged with respect to the capture reagent itself or to acharged substance conjugated to the capture reagent. As yet anotheralternative, the receptor molecule can be any specific binding memberwhich is immobilized upon (attached to) the solid phase and which hasthe ability to immobilize the capture reagent through a specific bindingreaction. The receptor molecule enables the indirect binding of thecapture reagent to a solid phase material before the performance of theassay or during the performance of the assay. The solid phase thus canbe a plastic, derivatized plastic, magnetic or non-magnetic metal, glassor silicon surface of a test tube, microliter well, sheet, bead,microparticle, chip, and other configurations known to those of ordinaryskill in the art.

It is contemplated and within the scope of the invention that the solidphase also can comprise any suitable porous material with sufficientporosity to allow access by detection antibodies and a suitable surfaceaffinity to bind antigens. Microporous structures are generallypreferred, but materials with gel structure in the hydrated state may beused as well. Such useful solid supports include:

natural polymeric carbohydrates and their synthetically modified,cross-linked or substituted derivatives, such as agar, agarose,cross-linked alginic acid, substituted and cross-linked guar gums,cellulose esters, especially with nitric acid and carboxylic acids,mixed cellulose esters, and cellulose ethers; natural polymerscontaining nitrogen, such as proteins and derivatives, includingcross-linked or modified gelatins; natural hydrocarbon polymers, such aslatex and rubber; synthetic polymers which may be prepared with suitablyporous structures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer. All of these materials maybe used in suitable shapes, such as films, sheets, or plates, or theymay be coated onto or bonded or laminated to appropriate inert carriers,such as paper, glass, plastic films, or fabrics.

The porous structure of nitrocellulose has excellent absorption andadsorption qualities for a wide variety of reagents including monoclonalantibodies. Nylon also possesses similar characteristics and also issuitable.

It is contemplated that such porous solid supports described hereinaboveare preferably in the form of sheets of thickness from about 0.01 to 0.5mm, preferably about 0.1 mm. The pore size may vary within wide limits,and is preferably from about 0.025 to 15 microns, especially from about0.15 to 15 microns.

To change or enhance the intrinsic charge of the solid phase, a chargedsubstance can be coated directly to the material or onto microparticleswhich then are retained by a solid phase support material.Alternatively, microparticles can serve as the solid phase, by beingretained in a column or being suspended in the mixture of solublereagents and test sample, or the particles themselves can be retainedand immobilized by a solid phase support material. By "retained andimmobilized" is meant that the particles on or in the support materialare not capable of substantial movement to positions elsewhere withinthe support material. The particles can be selected by one skilled inthe art from any suitable type of particulate material and include thosecomposed of polystyrene, polymethylacrylate, polypropylene, latex,polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similarmaterials. The size of the particles is not critical, although it ispreferred that the average diameter of the particles be smaller than theaverage pore size of the support material being used. Thus, embodimentswhich utilize various other solid phases also are contemplated and arewithin the scope of this invention. For example, ion capture proceduresfor immobilizing an immobilizable reaction complex with a negativelycharged polymer, described in co-pending U.S. patent application Ser.No. 150,278 corresponding to EP Publication No. 0326100, and U.S. patentapplication Ser. No. 375,029 (EP Publication No. 0406473), can beemployed according to the present invention to effect a fastsolution-phase immunochemical reaction. An immobilizable immune complexis separated from the rest of the reaction mixture by ionic interactionsbetween the negatively charged polyanion/immune complex and thepreviously treated, positively charged porous matrix and detected byusing various signal generating systems previously described, includingthose described in chemiluminescent signal measurements as described inco-pending U.S. patent application Ser. No. 921,979 corresponding to EPOPublication No. 0 273,115.

Also, the methods of the present invention can be adapted for use insystems which utilize microparticle technology including automated andsemi-automated systems wherein the solid phase comprises amicroparticle. Such systems include those described in pending U.S.patent application Ser. No. 425,651 and U.S. Pat. No. 5,089,424, whichcorrespond to published EPO applications Nos. EP O 425 633 and EP O 424634, respectively, and U.S. Pat. No. 5,006,309.

The indicator reagent comprises a signal generating compound (label)which is capable of generating a measurable signal detectable byexternal means conjugated (attached) to a specific binding member forHEV. "Specific binding member" as used herein means a member of aspecific binding pair. That is, two different molecules where one of themolecules through chemical or physical means specifically binds to thesecond molecule. In addition to being an antibody member of a specificbinding pair for HEV, the indicator reagent also can be a member of anyspecific binding pair, including either hapten-anti-hapten systems suchas biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin,a complementary nucleotide sequence, an effector or a receptor molecule,an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, andthe like. An immunoreactive specific binding member can be an antibody,an antigen, or an antibody/antigen complex that is capable of bindingeither to HEV as in a sandwich assay, to the capture reagent as in acompetitive assay, or to the ancillary specific binding member as in anindirect assay.

The various signal generating compounds (labels) contemplated includechromogens, catalysts such as enzymes, luminescent compounds such asfluorescein and rhodamine, chemiluminescent compounds, radioactiveelements, and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase, and the like.The selection of a particular label is not critical, but it will becapable of producing a signal either by itself or in conjunction withone or more additional substances.

Other embodiments which utilize various other solid phases also arecontemplated and are within the scope of this invention. For example,ion capture procedures for immobilizing an immobilizable reactioncomplex with a negatively charged polymer, described in co-pending U.S.patent application Ser. No. 150,278 corresponding to EP publication0326100, and U.S. patent application Ser. No. 375,029 (EP publicationno. 0406473) both of which enjoy common ownership and are incorporatedherein by reference, can be employed according to the present inventionto effect a fast solution-phase immunochemical reaction. Animmobilizable immune complex is separated from the rest of the reactionmixture by ionic interactions between the negatively chargedpoly-anion/immune complex and the previously treated, positively chargedporous matrix and detected by using various signal generating systemspreviously described, including those described in chemiluminescentsignal measurements as described in co-pending U.S. patent applicationSer. No. 921,979 corresponding to EPO Publication No. 0 273,115, whichenjoys common ownership and which is incorporated herein by reference.

Also, the methods of the present invention can be adapted for use insystems which utilize microparticle technology including in automatedand semi-automated systems wherein the solid phase comprises amicroparticle. Such systems include those described in pending U.S.patent applications 425,651 and 425,643, which correspond to publishedEPO applications Nos. EP 0 425 633 and EP 0 424 634, respectively, whichare incorporated herein by reference.

The use of scanning probe microscopy (SPM) for immunoassays also is atechnology to which the monoclonal antibodies of the present inventionare easily adaptable. In scanning probe microscopy, in particular inatomic force microscopy, the capture phase, for example, at least one ofthe monoclonal antibodies of the invention, is adhered to a solid phaseand a scanning probe microscope is utilized to detect antigen/antibodycomplexes which may be present on the surface of the solid phase. Theuse of scanning tunneling microscopy eliminates the need for labelswhich normally must be utilized in many immunoassay systems to detectantigen/antibody complexes. Such a system is described in pending U.S.patent application Ser. No. 662,147, which enjoys common ownership andis incorporated herein by reference.

The use of SPM to monitor specific binding reactions can occur in manyways. In one embodiment, one member of a specific binding partner(analyte specific substance which is the monoclonal antibody of theinvention) is attached to a surface suitable for scanning. Theattachment of the analyte specific substance may be by adsorption to atest piece which comprises a solid phase of a plastic or metal surface,following methods known to those of ordinary skill in the art. Or,covalent attachment of a specific binding partner (analyte specificsubstance) to a test piece which test piece comprises a solid phase ofderivatized plastic, metal, silicon, or glass may be utilized. Covalentattachment methods are known to those skilled in the art and include avariety of means to irreversibly link specific binding partners to thetest piece. If the test piece is silicon or glass, the surface must beactivated prior to attaching the specific binding partner. Activatedsilane compounds such as triethoxy amino propyl silane (available fromSigma Chemical Co., St. Louis, Mo.), triethoxy vinyl silane (AldrichChemical Co., Milwaukee, Wisc.), and (3-mercapto-propyl)-trimethoxysilane (Sigma Chemical Co., St. Louis, Mo.) can be used to introducereactive groups such as amino-, vinyl, and thiol, respectively. Suchactivated surfaces can be used to link the binding partner directly (inthe cases of amino or thiol) or the activated surface can be furtherreacted with linkers such as glutaraldehyde, bis (succinimidyl)suberate, SPPD 9 succinimidyl 3-[2-pyridyldithio]propionate), SMCC(succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate), SIAB(succinimidyl [4-iodoacetyl]aminobenzoate), and SMPB (succinimidyl4-[1-maleimidophenyl]butyrate) to separate the binding partner from thesurface. The vinyl group can be oxidized to provide a means for covalentattachment. It also can be used as an anchor for the polymerization ofvarious polymers such as poly acrylic acid, which can provide multipleattachment points for specific binding partners. The amino surface canbe reacted with oxidized dextrans of various molecular weights toprovide hydrophilic linkers of different size and capacity. Examples ofoxidizable dextrans include Dextran T-40 (molecular weight 40,000daltons), Dextran T-110 (molecular weight 110,000 daltons), DextranT-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight2,000,000 daltons) (all of which are available from Pharmacia,Piscataway, N.J.), or Ficoll (molecular weight 70,000 daltons (availablefrom Sigma Chemical Co., St. Louis, Mo.). Also, polyelectrolyteinteractions may be used to immobilize a specific binding partner on asurface of a test piece by using techniques and chemistries described bypending U.S. patent applications Ser. No. 150,278, filed Jan. 29, 1988,and Ser. No. 375,029, filed Jul. 7, 1989, each of which enjoys commonownership and each of which is incorporated herein by reference. Thepreferred method of attachment is by covalent means. Followingattachment of a specific binding member, the surface may be furthertreated with materials such as serum, proteins, or other blocking agentsto minimize non-specific binding. The surface also may be scanned eitherat the site of manufacture or point of use to verify its suitability forassay purposes. The scanning process is not anticipated to alter thespecific binding properties of the test piece.

While the present invention discloses the preference for the use ofsolid phases, it is contemplated that the monoclonal antibody of thepresent invention can be utilized in non-solid phase assay systems.These assay systems are known to those skilled in the art, and areconsidered to be within the scope of the present invention.

The monoclonal antibody of the invention can be used as a positivecontrol in an assay which is designed to detect the presence of HEVantibody. In an assay which detected the presence of HEV antibody in atest sample, HEV antigens would be used as a capture phase. These HEVantigens could be prepared by various means from vital lysates,synthetic peptides of various immunogenic regions of the HEV genome,and/or recombinant proteins produced by using either synthetic or nativeantigens or epitopes of antigens. It also is contemplated that thesetypes of HEV antigens could be employed in a variety of assay formatsincluding those described herein as either the capture phase ordetection phase. The use of the monoclonal antibody of the inventionwould ensure that the reagents provided to detect HEV antibody wereperforming adequately by being used in place of a test serum in theperformance of the assay, according to procedures known to those ofordinary skill in the art.

It is contemplated that the reagent employed for the assay can beprovided in the form of a kit with one or more containers such as vialsor bottles, with each container containing a separate reagent such as amonoclonal antibody, or a cocktail of monoclonal antibodies, employed inthe assay. These kits also could contain vials or containers of otherreagents needed for performing the assay, such as washing, processingand indicator reagents.

The following examples demonstrate the advantages and utility of theH67C46 monoclonal antibody of the invention by describing methods forthe development, characterization, epitope mapping and clinical utilityof the monoclonal antibodies. The methods used for monoclonal antibodydevelopment follow procedures known in the art and detailed in Kohlerand Milstein, Nature 256:494 (1975), and reviewed in J. G. R. Hurrel,ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRCPress, Inc., Boco Raton, Fla. (1982).

To practice the invention, a recombinant DNA clone was constructed tocontain the gene encoding the full length orf-3 of HEV as a fusionprotein with CKS. The antigen (designated 8-5 protein) was used toimmunize a mouse from which an immune splenocyte was fused to aSP2/0-Ag14 myeloma cell to produce a hybridoma cell line that secretes amonoclonal antibody of immunoglobulin (Ig) class G₁ (IgG₁) reactive withHEV orf-3. The resultant immunoglobulin was produced in mouse ascitesfluid and was purified by affinity chromatography.

The examples set forth below are meant to illustrate, but not to limit,the spirit and scope of the invention.

EXAMPLE 1 Generation and Characterization of Hybridoma

A. Production of recombinant HEV Protein.

Plasmid pGEX-HEV-ORF3-8-5 (FIG. 1) was obtained from Genelabs, Inc.,Redwood City, Calif. and encodes the complete 123 amino acids of orf-3antigen of HEV as a glutathione S-transferase (GST) fusion in the pGEXexpression system. The gene encoding the full length HEV orf-3,designated as the 8-5 protein, (see P. O. Yarbough, et al., supra) wascloned and expressed as a chimeric fusion protein with CMP-KDOsynthetase (CKS) in plasmid pJ0201 in E. coli according to methods knownin the art. The CKS/HEV-8-5 protein was expressed in E. coli strainXL1-Blue at greater than 20% of total cell protein after induction withisopropyl b-D thiogalactoside (IPTG). The clones were propagated in 10liter fermentors, yielding 170-260 grams of wet cell paste perfermentation.

B. CKS-HEV Protein Solubilization.

The E. coli cells expressing the 8-5 protein were lysed at pH 10 in thepresence of various protease inhibitors. The CKS fusion protein wasinsoluble and remained in the pellet after centrifugation. Pellets werewashed with various detergents to remove non-specific proteins.Following solubilization in Tris, pH 8.5 containing 0.5% SDS, the CKSfusion protein was found to represent 50 to 60% of the total protein asevaluated by SDS-PAGE and gel densitometry. The solubilized protein wasfurther purified by Sephacryl S-300HR column chromatography. Fractionswere analyzed by SDS-PAGE, pooled and evaluated for purity by geldensitometry. Final purified protein was greater than 90% pure.

C. Immunization of Mice

The immunization regimen (4 mice) consisted of an initial immunizationwith additional immunizations at six and nine weeks. For the primaryimmunization, 10 μg CKS-HEV orf-3 8-5 extracted and solubilized(unpurified) recombinant protein, prepared as described above, wasemulsified in complete Freund's adjuvant. Four mice (Balb/c) wereinoculated intraperitoneally with this emulsion. At three weekspost-immunization, mouse serum was screened for enzyme immunoassay (EIA)immunoreactivity as described below. The serum anti-HEV titer testedagainst beadcoated with HEV 8-5 was 10⁶ in specimen diluent with no E.coil or CKS lysate added and 10⁵ in specimen diluent containing E. coiland CKS lysate, thus indicating only a 10-fold reactivity tonon-specific proteins. The mouse serum antibody titer against beadscoated with HEV orf-3 synthetic peptide spB4-2, prepared as described incopending U.S. patent application Ser. No. 07/089,877 filed Jul. 9, 1993was 5×10³, indicating reactivity to the carboxyl terminus of the HEVorf-3 protein.

Six weeks after the first immunization, two mice were boostedintraperitoneally with 10 μg of the immunizing protein in incompleteFreund's adjuvant. At week nine, post-immunization anti-HEV titersagainst beads coated with protein HEV 8-5 described above were 6.25×10⁶in specimen diluent with no additives to block out unwanted reactivity,2×10⁶ in specimen diluent with additives and 6.25×10⁶ against spB4-2.This mouse was then boosted intravenously (via the tail vein) with 30 μgof the immunizing protein and used for fusion 3 days later.

D. Establishment of the Hybridomas

Normal mouse splenocytes were prepared by aseptically removing thespleen from a non-immunized mouse and crushing it using the plunger of asyringe through a screen that fits into a 60×15 mm Petri dish containinga small amount of Dulbecco's Minimum Essential (DME) medium. Thesplenocyte solution was washed with DME medium, and the red blood cellswere lysed by adding 1 ml of 0.83% NH₄ Cl in 10mM Tris to the cellpellet for 1 minute. The cells were again washed and resuspended in 40mls of DME medium containing 20% Fetal Bovine Serum (FBS).

On the day of the fusion, the immunized mouse was sacrificed, and thespleen was removed and processed as described herein for the preparationof normal splenocytes except that the splenocytes were resuspended in 10mls DME medium and counted. Splenocytes were fused in a 1:1 ratio withthe SP2/0 myeloma cell line using the described modification ofconventional methods (Kohler and Milstein, supra). Cells were plated at1×10⁶ splenocytes per well of a 24 multi-well tissue culture tray in 1.5ml DME medium containing 20% FBS, hypoxanthine, aminopterin, andthymidine (HAT). After 24 hours. 200,000 normal splenocytes were addedper 24 multi well (0.5 ml). HAT medium was added on day 5 and replacedon day 7.

Four of ninety initial hybrids from the fusion were positive forantibodies to HEV. Hybrid #67 (H67) had an optical density (O.D). of0.988 when tested at a 1:100 dilution against 8-5 antigen-coated beads,was negative against CKS-only coated beads, and had an O.D. of 1.498 ata 1:100 dilution against HEV synthetic antigen spB4-2 beads. H67 wasthen cloned at one cell per well by limiting dilution technique asdescribed in Goding, Monoclonal Antibodies: Principles and Practices,2nd ed, Academic Press, New York [1986).

E. Establishment Of Clones By Limiting Dilution

The viable cells in the antibody positive wells were counted, and foreach 96 multiwell tissue culture tray, an aliquot containing 100 cellswas added to 20 mls of DME media containing 20% Fetal Bovine Serum (FBS)and 5×10⁶ normal mouse splenocytes. An eight channel piperman adjustedto 0.2 ml was used to plant the cell suspension. The trays wereincubated at 37° C. in a humidified 5% CO₂ incubator and refed on days 5and 7 or as needed because of evaporation. When the wells containinggrowth were 30-50% confluent (usually between 10-21 days), those wellscontaining only 1 colony were sampled and tested for antibody activity.Several positive wells from each hybrid were selected and the cells wereexpanded. A single clone designated H67C46 resulted which had reactivityagainst HEV orf-3(8-5), and in particular, to the spB4-2 carboxyterminal region.

EXAMPLE 2

Production and Purification of the H67C46 Monoclonal Antibody

Clones selected for further evaluation were scaled up in tissue cultureT-flasks and 10⁶ cells were injected into the peritoneal cavity ofpre-pristaned BALB/c mice (Charles River Biotechnical Services, Inc.,Wilmington, Mass.) (see Hurrell, supra). The resulting ascites fluid washarvested 7-10 days after injection, centrifuged and frozen. Theantibody was affinity purified on a Protein A Sepharose CL-4B(Pharmacia-LKB Biotechnologies, Piscataway, N.J.). Bound monoclonalantibody was eluted from the column at a pH of 5.5, indicating itssubtype as IgG₁.

EXAMPLE 3

Screening and Characterization of H67C46 Monoclonal Antibody

To screen and characterize the antibody, enzyme immunoassays (EIA's)were utilized which contained different antigens on the solid phase(polystyrene beads): the CKS-HEV recombinant orf-3 (8-5) (prepared asdescribed in Example 1) was coated on the solid phase in either theunpurified (extracted and solubilized) form (ca. 50-60% pure HEVprotein) or a purified protein (>90% pure), another EIA used thesynthetic peptide spB4-2 on the solid phase, and to detect non-specificbinding the solid phase was coated with CKS alone or a non-HEV CKSrecombinant protein.

For all assays, mouse serum, tissue culture supernatant, mouse ascitesfluid samples or monoclonal antibody (MAb) H67C46 obtained above wereserially diluted 5 to 10-fold in specimen diluent containingphosphate-buffered-saline/Tris/EDTA, pH 7.8 with 20% goat serum, 10%fetal calf serum, 1% bovine serum albumin, 0.2% Triton-X 100, 0.1%sodium azide, with or without 1% E. coli lysate and 0.01% CKS added,then 200 μl was incubated with each solid phase for 1-2 hours at 40° C.Beads were then washed 3 times with 5 ml distilled water. Bound antibodywas detected using 200 μl HRPO-labeled second antibody (Goat anti-mouseIgG H+L) from Kirkegaard and Perry Labs) at 0.3 μg/mL in conjugatediluent containing Tris-buffered-saline with 10% goat serum, 10% fetalcalf serum and 0.15% Triton-X 100. Beads were incubated with conjugatefor 1 hour at 40° C., and washed as before. O-Phenylenediamine (OPD)substrate was freshly prepared by adding 1 OPD tablet (Abbott) per 5 mlsof OPD Diluent (Abbott), and 300 μl of OPD substrate solution was thenadded to each washed bead and incubated in the dark at room temperaturefor 30 minutes. One ml of sulfuric acid added to stop the reaction, andthe amount of color generated was determined by measuring the absorbanceof the substrate at 492 nm within 2 hours of sulfuric acid addition.Results given in Table 1 below indicate that the antibody was specificfor the orf-3 region of HEV.

                  TABLE I                                                         ______________________________________                                        Characterization of H67C46 Monoclonal Antibody                                       Absorbance at 492 nm                                                   Dilution (-1)                                                                          Recombinant HEV                                                                            Recombinant HEV synthetic                               Clone H67C46                                                                           8-5-coated bead                                                                            CKS coated bead                                                                           peptide spB4-2                              ______________________________________                                        10       >2           0.042       >2                                          1 × 10.sup.3                                                                     >2           0.018       >2                                          10 × 10.sup.3                                                                    >2           0.012       >2                                          100 × 10.sup.3                                                                   >2           0.016       >2                                          1 × 10.sup.6                                                                     0.636        0.013       0.685                                       ______________________________________                                    

EXAMPLE 4

Competitive Assay for Antibody to HEV:

Solid phase was coated with HEV orf-3 (8-5) or a combination of proteinscoding for the orf-3 and orf-2 regions. The solid phases werepre-incubated with a 1:2 dilution of HEV negative human plasma (negativecontrol) or human plasma known to contain HEV Ab (sample) in specimendiluent overnight at room temperature. Beads were then washed andmonoclonal antibody (MAb) of the invention against HEV orf-3, diluted togive an O.D. of 1.000-1.500 when no inhibitor is present, was added tothe appropriate beads and incubated for 1 hour at 40° C. The beads werethen washed and HRPO-goat anti-mouse IgG (H+L) was added and incubatedfor 2 hours at 40° C. The beads were washed and label detection wasperformed using OPD substrate as described above.

Theoretically, a positive HEV Ab containing sample should reduce thesignal 50%. In the example provided the HEV Ab positive sample showed57.6% inhibition of MAb H67C46 when the 8-5 bead was used, and 35.3%inhibition when the HEV orf-2/orf-3 Combo bead was used. The results aregiven in Table II below.

                  TABLE II                                                        ______________________________________                                        Competitive Assay for Antibody to HEV orf-3                                             HEV BEAD                                                                      Recombinant Antigen for                                                                    Recombinant Antigens                                             orf-3        for orf-2 and orf-3                                              % Inhibition % Inhibition                                           ______________________________________                                        Pre-incubation with:                                                          Specimen Diluent                                                                          --             --                                                 Only                                                                          Negative Human                                                                            --             --                                                 Plasma                                                                        Human anti-HEV +                                                                          57.6           35.3                                               Plasma                                                                        ______________________________________                                    

EXAMPLE 5

Competitive Assay for orf-3 Antigen to HEV

Solid phase is coated with HEV orf-3 (8-5) protein or with spB4-2synthetic peptide. Monoclonal antibody (MAb) H67C46, at a concentrationpreviously determined to give an O.D. of 1.000-2.000 in this assayformat, is pre-incubated with sample at a time and temperature longenough to allow binding of HEV antigen in the sample to MAb (30-120minutes, at room temperature to 40° C.). The solid phase is then added,and the mixture and bead are incubated at a time and temperature longenough to allow any remaining unbound MAb to bind to the solid phase(temperature range: room temperature to 40° C., time range: 1-16 hours).Binding of MAb to the solid phase is then detected either using alabeled second antibody (e.g. horseradish peroxidase (HRPO)-labelledGoat anti-mouse IgG) with incubation of 1-2 hours at 40° C., or MAbitself could be labeled, thus making the assay potentially performablein one step. Label detection is then performed (i.e. if using HRPOlabeled Ab, add OPD substrate, incubate 30 minutes, add sulfuric acid tostop the reaction, and read at 492 nm). A positive, HEV antigencontaining sample will inhibit with MAb for binding to the solid phaseand reduce the signal at least 50%. Hybridoma cell line H67C46 wasdeposited with the American Type Culture Collection (A.T.C.C.), 12301Parklawn Drive, Rockville, Md. 20852, on Jan. 12, 1994, and accordedA.T.C.C. Deposit Accession Number HB 11522.

What is claimed is:
 1. A monoclonal antibody or fragment thereof whichspecifically binds to hepatitis E virus (HEV) orf-3-encoded protein,wherein said monoclonal antibody is secreted by a cell line ATCC depositnumber HB
 11522. 2. A hybridoma cell line which secretes a monoclonalantibody which specifically binds to hepatitis E virus (HEV)orf-3-encoded protein, wherein said cell line is ATCC deposit number HB11522.
 3. A competitive assay method for determining the presence ofHepatitis E Virus (HEV) antibody which may be present in a test sample,comprising:a. contacting a test sample suspected of containing HEVantibodies with a solid phase coated with at least one HEV orf-3 antigenand an indicator reagent comprising a signal generating compound whichgenerates a measurable signal and a monoclonal antibody whichspecifically binds to said HEV orf-3 antigen, for a time and underconditions sufficient to form antigen/antibody complexes of the testsample and solid phase and/or indicator reagent and solid phase; and b.determining the presence of HEV antibody present in the test sample bydetecting the reduction in binding of the indicator reagent to the solidphase as compared to the signal generated from a negative test sample toindicate the presence of HEV antibody in the test sample, wherein saidmonoclonal antibody which specifically binds to said HEV orf-3 antigenis secreted by the cell line ATCC deposit number HB
 11522. 4. The methodof claim 3 wherein the signal generating compound is selected from thegroup consisting of a luminescent compound, a chemiluminescent compound,an enzyme and a radioactive element.
 5. The method of claim 4 whereinsaid enzyme is selected from the group consisting of horseradishperoxidase, alkaline phosphatase and beta-galactosidase.
 6. An assay kitfor determining the presence of HEV antigen or antibody in a test samplecomprising a container containing at least one monoclonal antibody orfragment thereof which specifically binds to HEV orf-3 antigen, whereinsaid monoclonal antibody is secreted by cell line ATCC deposit number HB11522.
 7. An immunoassay for detecting hepatitis E virus (HEV) antigenwhich may be present in a test sample comprising (a) contacting saidtest sample suspected of containing HEV antigen with a solid phase towhich an HEV specific antibody is attached to form an antigen-antibodycomplex, (b) contacting said complex with an indicator reagentcomprising an antibody attached to a label capable of generating adetectable signal wherein said indicator reagent specifically binds tosaid HEV antigen and (c) detecting the presence of HEV antigen in saidtest sample by detecting the signal generated by said indicator reagent,wherein the improvement comprises utilizing in either the solid phase ofstep (a) or the indicator reagent of step (b) a monoclonal antibody orfragment thereof, said monoclonal antibody secreted by the cell lineATCC deposit number HB 11522, wherein said monoclonal antibody orfragment thereof specifically binds to HEV orf-3 antigen.